Abstract

TRAP1 is an antiapoptotic heat shock protein, identified through an mRNA-differential display analysis in oxidants- adapted osteosarcoma cells. The main objective of my PhD thesis has been the characterization of the interaction between TRAP1 and TBP7 (S6-ATPase 4/Rpt3), a component of the 19S proteasome regulatory subunit, one of the putative TRAP1 “partners” identified by our LC-MS/MS analysis. I demonstrated that TRAP1 and TBP7 are located in the endoplasmic reticulum (ER), on the outer side of this compartment; they directly interact in the ER, as demonstrated by coimmunoprecipitation experiments and by FRET analysis. The information available on the TRAP1 pathway describes just a few well-characterized functions of this protein in mitochondria. This is the first demonstration of TRAP1’s presence in this cellular compartment. Given the ER localization of TRAP1 and TBP7, the involvement of these two proteins in ER homeostasis has been investigated. TRAP1silencing by short-hairpin RNAs, in cells exposed to thapsigargin-induced ER stress, correlates with upregulation of BiP/Grp78,thus suggesting a role of TRAP1 in the refolding of damaged proteins and in ER stress protection. Consistently, TRAP1 and/or TBP7 interference enhanced stress-induced cell death and increased intracellular protein ubiquitination. These experiments led us to hypothesize an involvement of TRAP1 in protein quality control for mistargeted/misfolded mitochondria-destined proteins, through interaction with the regulatory proteasome protein TBP7. Remarkably, expression of specific MITO proteins decreased upon TRAP1 interference as a consequence of increased ubiquitination. The proposed TRAP1 network has an impact in vivo, as it is conserved in human colorectal cancers, is controlled by ER-localized TRAP1 interacting with TBP7 and provides a novel model of the ER–mitochondria crosstalk.
The data described in my thesis on the functional characterization of TRAP1/TBP7 interaction allowed us to draw the following hypopthesis: when neo-synthesized proteins are damaged, they are not imported into mitochondria, but sequestered by TRAP1 to be refolded/ repaired; if this attempt fails, these substrates are ubiquitinated, recognised by the regulatory subunits of proteasome to which TBP7 belongs, and delivered to the proteolytic core for degradation.